DOMESTIC ANIMAL ENDOCRINOLOGY
LUTEINIZING HORMONE SECRETION IN HYPOPHYSIAL STALK-TRANSECTED GILTS GIVEN HYDROCORTISONE ACETATE AND PULSATILE GONADOTROPIN-RELEASING HORMONE 1 M.J. Estienne.2, C.R. Barb**, J.S. Kesner*.3, R.R. Kraeling** and G.B. Rampacek* *Animal and Dairy Science Department University of Georgia Athens, Georgia 30602 and **Animal Physiology Research Unit United States Department of Agriculture Agricultural Research Service Richard B. Russell Agricultural Research Center Athens, Georgia 30613 Received October 9, 1990
ABSTRACT The site within the hypothalamic-pituitary axis at which cortisol acts to inhibit luteinizing hormone (LH) secretion was investigated in female pigs. Six ovariectomized, hypophysial stalk-transected (HST) gilts were given 1 lag pulses of gonadotropin releasing-hormone (GnRH) iv every 45 min from day 0 to 12. On days 6-12, each of 3 gilts received either hydrocortisone acetate (HCA; 3.2 mg/kg body weight) or oil vehicle im at 12-hr intervals. Four ovariectomized, pituitary stalk-intact gilts served as controls and received HCA and pulses of 3.5% sodium citrate. Jugular blood was sampled dally and every 15 min for 5 hr on days 5 and 12. Treatment with HCA decreased serum LH concentrations and LH pulse frequency in stalk-intact animals. In contrast, serum LH concentrations, as well as the frequency and amplitude of LH pulses, were unaffected by HCA in HST gilts and were similar to those observed in oil-treated HST gilts. We suggest that chronically elevated concentrations of circulating cortisol inhibit LH secretion in pigs by acting at the level of the hypothalamus.
INTRODUCTION Stress reduces reproductive efficiency in several species presumably via activation of the hypothalamic-pituitary-adrenal axis (For review, see 1). The adrenal glands respond to stress with increased secretion of glucocorticoids (2), and these steroids inhibit luteinizing hormone (LH) release in several species including the pig (3-5). Thus, the etiology of stressinduced alterations in reproductive function could involve a suppression of gonadotropin secretion by glucocorticoids. Luteinizing hormone release from the anterior pituitary gland is pulsatile in ovariectomized (6) and ovary-intact pigs (6-8), presumably in response to episodic discharges of gonadotropin-releasing hormone (GnRH) from the hypothalamus. Pulsatile secretion of LH in pigs is abolished by hypophysial stalk-transection (HST; 9--11), hypothalamic deafferentation (12), immunization against GnRH (13), or by feeding the centrally active compound methallibure (14). Thus, cortisol may decrease LH secretion in the ovariectomized pig by inhibiting the secretion of GnRH and/or by acting directly on the pituitary gland. In other species, the glucocorticoids exert suppressive actions at both loci (1). Furthermore, in rhesus monkeys, glucocorticoids act at the gonadal level to block follicular secretion of estrogen and hence the preovulatory surge of LH (15). In an effort to determine sites within the hypothalamic-pituitary axis at which glucocorticoids may act to suppress gonadotropin release in swine, we assessed LH secretion after Copyright © 1991 Butterworth--Heinemann
ESTIENNE ET AL.
the administration of hydrocortisone acetate (HCA) to ovariectomized, HST gilts receiving unvarying pulses of GnRH.
MATERIALS AND METHODS Animals. Six prepuberal, crossbred gilts were subjected to HST using the supraorbital approach to hypophysectomy of du Mesnil du Buisson et al. (16) as modified by Kraeling (17). A Teflon disc was placed between the severed ends of the stalk to prevent vascular regeneration. Approximately 2 weeks following surgery, HST gilts and 4 contemporary pituitary stalk-intact gilts were ovariectomized. One of the stalk-intact gilts had underwent a sham-transection of the hypophysial stalk. The experiment was conducted 18 weeks following ovariectomy when average age and body weight of gilts were 9.6 months and 136.7 kg, respectively. Before and during the investigation, animals were individually penned in an environmentally controlled building (temperature= 20 C; photoperiod= 12 hr light, 12 hr dark). Gilts received 1.8 kg of a corn-soybean meal diet daily (0600 hr) and were allowed water ad libitum.
Correct placement of the Teflon barrier in HST gilts was confirmed at necropsy (14 weeks after the experiment), at which time body, whole pituitary, and right adrenal gland weights for all gilts were recorded. General. On the day prior to the experiment, a catheter was inserted into a jugular vein (18) for sampling blood and infusing GnRH (GnRH acetate salt; Sigma Chemical Co., St. Louis, MO). The mechanics for delivering GnRH and basis for selecting the dose have been described previously (1 l, 14, 19). Briefly, 1 Ixg GnRH was dissolved in 3.5% sodium citrate and was infused during a 5-min pulse every 45 min by peristaltic pumps (Ismatec; ColeParmer, Chicago, IL) driven by a programmable timer (Chrontrol; Lindburg Enterprises, San Diego, CA). This GnRH dose, when given to ovariectomized gilts in which endogenous GnRH release had been blocked with methallibure, restored LH pulse amplitude and frequency to levels similar to those observed in untreated ovariectomized gilts (14). In HST gilts, this dose sustained LH secretion for 12 d (19) and did not evoke maximal LH release (11). Hydrocortisone 21-Acetate (HCA; Sigma Chemical Co.) was dissolved in corn oil and injected im (3.2 mg/kg body weight) every 12 hr (0700 and 1900 hr). This treatment regimen was previously shown to inhibit LH secretion in gilts (5). Experimental Design. All HST gilts were given pulses of GnRH from day 0 (1625 hr) to day 12 (1230 hr). From day 6 to day 12, each of 3 HST gilts received HCA or vehicle injections. The four stalk-intact gilts served as controls and received HCA and pulses of 3.5% sodium citrate. The HCA treatment was successful in elevating circulating levels of cortisol. Serum cortisol concentrations prior to treatment with HCA or oil on day 5 were similar between groups and averaged 20 _+4.1 ng/ml across treatments. Cortisol concentrations on day 12 in stalk-intact and HST animals receiving HCA, and HST gilts receiving oil were 152 _+9.6, 144 +_ 32.9, and 25.3 _+ 11.7 ng/ml, respectively (Figure 2). Blood samples were collected immediately before beginning (day 0) and 5 hr after stopping (day 12) GnRH treatment to verify that LH secretion in HST gilts was low in the absence of exogenous GnRH stimulation. Single blood samples were also collected daily (1200 hr) from day 1 to 12, immediately following a GnRH or vehicle pulse. Finally, pulsatile release of LH was assessed by sampling blood at 15-min intervals for 5 hr (0800-1300 hr) on days 5 and 12. Blood-handling procedures and assays. Blood samples were allowed to clot at 4 C for 24 hr, serum was harvested and then stored at -20 C until assayed. Concentrations of LH were determined by duplicate measurement of 300 ~1 aliquots of all samples using radioimmunoassay procedures previously described (14, 18, 20). The intraassay and interassay co-
LH AFTER HYDROCORTISONE ACETATE AND GNRH
efficients of variation were 7.7% and 9.3%, respectively. Assay sensitivity averaged 0.1 ng/ ml. Cortisol concentrations in single samples collected on days 5 (1400 h) and 12 (0800 hr) were determined by duplicate radioimmunoassay (21) of 100 [tl aliquots. The intraassay coefficient of variation and assay sensitivity were 5.1% and 0.1 ng/ml, respectively. Statistical Analyses. For each gilt, mean serum LH concentrations and the number and amplitude of LH pulses on day 5 and day 12 were determined. These data were subjected to analysis of variance for a repeated measures design (22) using a model that included treatment (i.e., HST plus HCA, HST plus oil, stalk-intact plus HCA), gilt within treatment, day, and treatment x day interaction as possible sources of variation. The effect of treatment was tested using animal within treatment as the error tenn. Day and treatment x day were tested against animal within treatment x day. If significant treatment x day interactions were detected, then additional analyses were conducted. One-way analyses of variance were conducted to determine the main effect of day within each treatment and the main effect of treatment within each day. Finally, specific comparisons between means were evaluated using Tukey's studentized range test (23). A LH pulse was defined as an increment exceeding a single standard deviation of the mean for LH concentration of a serum pool which was analyzed eight times in each assay (14, 19). Pulse amplitude equalled the difference between the pulse peak and the preceding nadir (14, 19). Pulse frequency was expressed as the number of pulses observed per hr. Concentrations of LH in daily blood samples were analyzed using a split-plot-in-time analysis of variance (22). The statistical model included treatment, day, and the treatment x day interaction as possible sources of variation. The error terms used for testing main effects and interactions, and the analyses performed when significant interactions were detected, were similar to those described above. Effects of day within treatments were also analyzed for significant linear and quadratic components. Body and endocrine gland weights for HST and stalk-intact gilts were compared using one way analysis of variance followed by Tukey's studentized range test (23). RESULTS Examination of the hypothalamo-hypophysial region of the HST gilts at necropsy revealed that the hypophysial stalk was completely severed and that the Teflon harrier had prevented vascular regeneration between the median eminence and the anterior pituitary gland. Whole pituitary (P<0.05) and right adrenal gland weights (P<0.1) were greater for intact than for HST gilts (Table 1). Before initiating GnRH replacement on day 0, serum LH concentrations in HST gilts were 0.17 _+0.01 ng/ml (mean _+ SE; Figure 1A and B). Pulsatile GnRH replacement progressively stimulated LH secretion in HST gilts, raising serum LH concentrations to 0.51 _+ 0.05 ng/ml after 5 d of treatment. Concentrations of LH in HST gilts decreased to 0.26 _+ 0.02 ng/ml following cessation of GnRH treatment on day 12 (data not shown).
TABLE l. BODY ANDENDOCRINEGLANDWEIGHTS(MEAN± SE) AT NECROPSYFOR HYPOPltYSIALSTALK-TRANSECTED(HST) AND PITUITARYSTALK-INTACTGILTSTHATRECEIVEDHYDROCORTISONEACETATE(HCA) OR On.
R. Adrenal Gland
HST + HCA
156 ± 14'
165 ± 1'
1.6 ± 0.2'
HST + Oil
167 ± 13'
174 ± 2"
2.1 ± 0.4'
Stalk-intact + HCA
157 ± 6'
414 ± 26b
3.4 ± 0.5 b
"bMe,ans in the same column with different superscripts differ (P
ESTIENNE ET AL.
0.6 0.4 0.2 A 0.0
E 0.8 2 tl)
E Un E
0.4 0.2 B 0.0
Sodium Citrate I
2.0 1.5 1.0 0.5 C Stalk-intact 0.0
Figure 1. Daily serum LH concentrations for HST gilts receiving GnRH and oil (A) or HCA (B), and pituitary stalk-intact gilts given 3.5% sodium citrate and HCA (C). Values are means + SE (n--4 for stalk-intact gilts, n=3 for each HST group), First data point represents samples collected before GnRH or 3.5% sodium citrate infusions began. All other samples were collected immediately after a GnRH or vehicle pulse. Note different ordinate scales. A treatment x day interaction (P<0.0001) was detected. Serum LH concentrations in HST gilts were unaffected (P>0.1) by oil or HCA treatment. In contrast, stalk-intact gilts receiving HCA exhibited a linear decrease (P<0.01) in serum LH concentrations.
A treatment x day interaction (P<0.000 I) was detected for daily serum LH concentrations (Figure 1). Serum LH concentrations in HST gilts were unaffected (P>0.1) by HCA or oil treatment (Figures 1A and B). Stalk-intact gilts receiving HCA, however, exhibited a linear decrease (P<0.01) in serum LH concentrations (Figure 1C). Depicted in Figure 2 are the serum cortisol concentrations, mean serum LH concentrations, LH pulse frequency and LH pulse amplitude on days 5 and 12 for each treatment group. Treatment x day interactions were detected for mean serum LH concentrations
LH AFTER H Y D R O C O R T I S O N E ACETATE AND GNRH
..-., 200 E ~
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0.0 0.6 W
r##/#~ 11111, ffJffJ
1.5 E 2
-~ 1.0 T
Stalk-intact + citrate + HCA
HST + GnRH + HCA
HST + GnRH + Oil
Figure 2. Serum cortisol concentrations, mean serum LH concentrations, LH pulse frequency and LH pulse amplitud¢ on day 5 (striped bars) and day 12 (open bars) for stalk-intact gilts givvn 3.5% sodium citrate and HCA and HST gilts receiving GnRH and HCA or oil. HCA and oil injections began on day 6. Values are means :1: SE (n--4 for stalk-intact gilts, nffi3 for each HST group). Treatment x day interactions were detected for re©an serum LH concentrations (P<0.001) and LH pulse frequency (P<0.03). These indices of LH secretion were decreasvd (brackets and * , P<0.07) by HCA treatment in stalk-intact gilts. In contrast, mean serum LH concentrations and LH pulse frequency in HST pigs were unaffected (P>0.1) by HCA or oil. A treatment effect (P<0.0007) existed for LH pulse amplitude as overall amplitude of LH pulses was greater (P<0.05) for stalkintact gilts compared with HST gilts.
ESTIENNE ET AL.
(P<0.001) and LH pulse frequency (P<0.03). These indices of LH secretion were decreased (P<0.07) by HCA treatment in stalk-intact gilts. In contrast, mean serum LH concentrations and LH pulse frequency in HST pigs were unaffected (P>0.1) by HCA or oil. A significant treatment effect (P<0.0007), but no treatment x day interaction (P>0.1) existed for LH pulse amplitude (Figure 2). Overall, pulse amplitude was greater (P<0.05) in stalk-intact gilts (0.45 + 0.05 ng/ml) compared to either HST treatment group. Amplitude of LH pulses was similar (P>0.1) for HST gilts receiving HCA (0.13 + 0.02 ng/ml) or oil (0.15 + 0.03 ng/ml). DISCUSSION Completeness of hypophysial stalk-transection and interruption of endogenous GnRH stimulation to the pituitary were confirmed by two approaches. First, the severed stalk and placement of the Teflon barrier were examined at necropsy. Secondly, serum LH concentrations were near the limits of detection for ovariectomized HST gilts in the absence of exogenous GnRH, providing endocrinologic evidence that hypothalamic inputs were not reaching the pituitary gland. The 1 gg pulses of GnRH effectively evoked pulses of LH in HST gilts, regardless of treatment. In absolute values, however, these pulses were generally of lower amplitude than those of stalk-intact gilts. This probably is a reflection of the smaller pituitary glands in HST compared with stalk-intact animals, as was previously described (24). Prior to steroid treatment, amplitude of LH pulses, expressed as a percentage of mean serum LH concentrations were 27 and 38% for HST and stalk-intact gilts, respectively. We have documented (11, 19, 25) that the GnRH replacement regimen employed in this investigation restores qualitative, if not quantitative, LH secretion in HST gilts. For example, ovary-intact, HST gilts receiving this treatment in addition to a single injection of pregnant mare serum gonadotropin (PMSG) display follicular growth and ovulate (25). In contrast, HST gilts receiving vehicle infusion fail to respond to PMSG (25). That cortisol concentrations (day 5) were similar for stalk-intact and HST gilts conforms to earlier observations in pigs (26) and sheep (27). Thus, the capability of the adrenal gland to secrete basal levels of cortisol remains intact in the absence of hypothalamic inputs. Chronically elevated cortisol concentrations decreased serum LH release in pituitary stalk-intact pigs, which is consistent with earlier work in gilts (4, 5) and boars (3). Release of LH in HST gilts in which GnRH was replaced, however, was unaffected by cortisol administration. Thus, we suggest that cortisol acts at a site other than the pituitary gland to inhibit gonadotropin secretion in swine. These results are consistent with the work of Fonda et al. (5), who demonstrated that HCA administration does not affect the response to GnRH in pituitary stalk-intact, ovariectomized gilts. Also, in vitro studies using dispersed pig pituitary cells demonstrated that only large, and potentially toxic, doses of cortisol (100 gg/ml) inhibited GnRH-induced LH release (28). The current study, however, examined only chronic effects of cortisol on LH secretion and does not preclude the possibility that acute elevations in circulating glucocorticoid concentrations may influence pituitary responsiveness to GnRH in the pig. Indeed, short-term elevations in plasma cortisol concentrations alter gonadotrope sensitivity to exogenously administered GnRH in prepubertal gilts (29) and boars (30). Dubey and Plant (31) suggested that glucocorticoids suppress GnRH secretion in monkeys. In the present experiment, HCA administration in stalk-intact gilts decreased not only mean serum LH concentrations but also LH pulse frequency, suggesting that the hypothalamic discharge of GnRH was inhibited. In earlier work (5), however, mean serum LH concentrations, but not LH pulse frequency, was decreased by HCA in ovariectomized gilts. The reason for the difference in the effectiveness of exogenous glucocorticoids to suppress LH pulse frequency is not readily apparent.
LH AFTER HYDROCORTISONE ACETATE AND GNRH
Results of the current investigation are consistent with the notion that HCA does not directly alter gonadotrope function. It is apparent, however, that the ability of glucocorticoids to alter gonadotropin secretion by direct action on the pituitary gland may vary with the species and sex of the animal studied as well as the steroid hormone milieu present. For example, glucocorticoids suppress gonadotrope responsiveness to GnRH in sheep (32) and cattle (33-35) but not monkeys (36). Glucocorticoids decrease the amount of LH secreted by female (37, 38), but not male (39) rat pituitary cells in vitro. Finally, exogenously administered cortisol inhibits GnRH-induced LH release in orchidectomized but not intact male rats (40). In summary, HCA administration decreased LH secretion in pituitary stalk-intact gilts, but not in HST gilts given unvarying pulses of GnRH. We suggest that cortisol inhibits LH secretion in ovariectomized pigs by acting at the level of the central nervous system. ACKNOWLEDGEMENTS/FOOTNOTES The authors wish W acknowledge Bennett Johnson and Elizabeth A. Price-Taras for their expert technical assistance. Address correspondence (including requests for reprints) to: Dr. G.B. Rampacek, Animal and Dairy Science Department, Livestock-Ponltry Building, University of Georgia, Athcns, GA 30602. ~Presentedin part at the Third InternationalConference on Pig Reproduction, April, 1989. University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, UK. This work was supported by USDA funds and State and Hatch funds allocated to the Georgia Agricultural Research Station. Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by the USDA or University of Georgia and does not imply its approval to the exclusion of other products that may be suitable. 2Prescnt Address: Department of Agriculnue, University of Maryland Eastern Shore, Princess Anne, MD 21853. 3Present Address: NIOSH-Experimental Toxicology Branch, Robert A. Taft Laboratories, 4676 Columbia Parkway, Mail Stop C-23, Cincinnati, OH 45226.
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the pituitary responsiveness to estradiol in the female rhesus monkey (Macaca mulatta). Biol Reprod 42:260-265, 1990. du Mesnil du Buisson F, Leglise PC, Chodkiewicz JP. Technique de l-hypophysectomie par voie transfrontale sus-orbitaire chez le porc. Ann Biol Anim Biochim Biophys 4:229-237, 1964. Kraeling RR. A modified supraorbital approach to hypophysectomy in the pig. Am J Vet Res 34:283-284, 1973. Kraeling RR, Rampacek GB, Cox NM, Kiser TE. Prolactin and luteinizing hormone secretion after bromocryptine (CB-154) treatment in lactating sows and ovariectomized gilts. J Anim Sci 54:1212-1220, 1982. Estienne MJ, Kesner JS, Kraeling RR, Rampacek GB, Barb CR. Luteinizing hormone secretion in hypophysial stalk-transected pigs given progesterone and pulsatile gonadotropin-releasing hormone. Proc Soc Exp Biol Med 190:14-17, 1989. Barb CR, Kraeling RR, Rampacek GB, Whisnant CS. Opioid inhibition stimulates luteinizing hormone and prolactin secretion in the gilt. Domest Anim Endocrinol 2:93-98, 1985. Fonda ES, Rampacek GB, Kraeling RR, Hart MA. Effect of storage time and temperature on steroid and protein hormone concentrations in porcine plasma and serum. Theriogenology 18:711-721, 1982. Gill JJ. Design and Analysis of Experiments in the Animal and Medical Sciences, Vol 2. Iowa State University Press, Ames, IA, 1978. Sokal RR, Rohlf FJ. Introduction to Biostatistics. WH Freeman and Co, San Francisco, CA, 1973. Kraeling RR, Barb CR, Rampacek GB. Hypophysectomy and hypophysial stalk transection in the pig: technique and application to studies of ovarian follicular development. In: Swine in Biomedical Research, Vol 1, Tumbleson ME (ed). Plenum Publishing Co, New York, p. 425--433, 1986. Kraeling RR, Kesner JS, Estienne MJ, Estienne CE, Barb CR, Rampacek GB. Follicle growth in hypophysial stalk-transected pigs given pulsatile GnRH and pregnant mare serum gonadotropin. Domest Anim Endocrinol 7:395-402, 1990. Estienne MJ, Kesner JS, Barb CR, Kraeling RR, Rampacek GB. On the site of action of naloxonestimulated cortisol secretion in gilts. Life Sci 43:161-166, 1988. Clarke IJ, Clements JA, Cummins JT, Dench F, Smith AI, Robinson PM, Funder JW. Elevated plasma levels of pro- opiomelanocortin-derived peptides in sheep following hypothalamo-pituitary disconnection. Neuroendocrinology 44:508-514, 1986. Li PS. Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro. Life Sci 41:2493-2501, 1987. Pearce GP, Paterson AM, Hughes PE. Effect of short-term elevations in plasma cortisol concentration on LH secretion in prepubertal gilts. J Reprod Fertil 83:413-418, 1988. Liptrap RM, Raeside JI. Effect of cortisol on the response to gonadotrophin releasing hormone in the boar. J Endocrinol 97:75-81, 1983. Dubey AK, Plant TM. A suppression of gonadotropin secretion by cortisol in castrated male rhesus monkey (Macaca mulatta) mediated by the interruption of hypothalamic gonadotropin-releasing hormone release. Biol Reprod 33:423-431, 1985. Matteri RL, Watson JG, Moberg GP. Stress or acute adrenocorticotrophin treatment suppresses LHRHinduced LH release in the ram. J Reprod Fertil 72:385-393, 1984. Li PS, Wagner WC. In vivo and in vitro studies on the effect of adrenocorticotropic hormone on the pituitary response to gonadotropin releasing hormone. Biol Reprod 29:25-37, 1983. Matteri RL, Moberg GP. Effect of cortisol or adrenocorticotrophin on release of luteinizing hormone induced by luteinizing hormone releasing hormone in the dairy heifer. J Endocrinol 92:141-146, 1982. Padmanabhan V, Keech C, Convey EM. Cortisol inhibits and adrenocorticotropin has no effect on luteinizing hormone- releasing hormone-induced release of luteinizing hormone from bovine pituitary cells in vitro. Endocrinology 112:1782-1787, 1983. Hayashi KT, Moberg GP. Influence of acute stress and the adrenal axis on regulation of LH and testosterone in the male rhesus monkey (Macaca mulatta). Am J Primatol 12:263-273, 1987. Suter DE, Schwartz NB. Effects of glucocorticoids on secretion of luteinizing hormone and folliclestimulating hormone by female rat pituitary cells in vitro. Endocrinology 117:849-854, 1985. Karnel F, Kubajak CL. Modulation of gonadotropin secretion by corticosterone: Interaction with gonadal steroids and mechanism of action. Endocrinology 121:561-568, 1987. Suter DE, Schwartz NB. Effects of glucocorticoids on responsiveness of luteinizing hormone and folliclestimulating hormone to gonadotropin-releasing hormone by male rat pituitary ceils in vitro. Endocrinology 117:855-859, 1985. Ringstrom SJ, Schwartz NB. Cortisol suppresses the LH, but not the FSH, response to gonadotropinreleasing hormone after orchidectomy. Endocrinology 116:472-474, 1985.